We have previously described a mouse IgG3 monoclonal antibody (AbR.sub.24) with a high degree of serological specificity for cultured human melanoma cells (1). All melanoma cell lines examined and two astrocytomas were positive for the heatstable cell surface antigen detected by this antibody. Although choroidal melanocytes and brain had low levels of the antigen, a wide variety of other cells and tissues were unreactive. Three other monoclonal antibodies (Abs C.sub.5, I.sub.24, and K.sub.9), having a similar restricted specificity, were derived from the same fusion. These antibodies showed the same strong reactivity with melanomas and lack of reactivity with epithelial cells, but had a slightly wider specificity range in that they also reacted weakly with MOLT-4 (a T-cell line), leukocytes and some fetal tissues.
The antigen detected by Ab R.sub.24 is identified herein as G.sub.D3- a previously characterized disialogangliodide. In comparison with other cells and tissues, melanomas have high levels of G.sub.D3. Thus, these antibodies are useful in determining whether a tissue sample is a melanoma or not. This is particularly important for characterizing lesions. These antibodies can also be used in determining concentrations of G.sub.D3 in serum, plasma, urine or other body fluids. This may aid in the early diagnosis of melanoma and possibly of other disorders where there are elevated glycolipid levels.
FIG. 1 time course for the reexpression of AbR.sub.24 -reactive antigen on SK-MEL-28 cells after neuraminidase treatment. Assay: PA-MHA.
FIG. 2 localization of AbR.sub.24 -reactive glycolipid on thin layer chromatography using glycolipid-mediated immune adherence (GMIA) assay. Acidic glycolipids from SK-MEL-28 cells were separated by TLC in solvent 1. Silica gel bands (1 cm wide) were scraped from the plate, extracted with C:M (1.2) and assayed for antigens by GMIA as described in the text.
FIG. 3 thin layer chromatography of acidic glycolipid fractions from a number of cell lines and tissues. Lane 1: G.sub.M3 ; 2: G.sub.D3 ; 3: G.sub.M1 ; 4: SK-MEL-28 melanoma cell line; 5: AbR.sub.24 -reactive antigen isolated from SK-MEL-28; 6: SK-MEL-37 melanoma cell line; 7: SK-MEL-64 melanoma cell line; 8: MeWo; 9: SK-MEL-13 melanoma cell line; 10: melanoma (surgical specimen); 11: MOLT-4 T-cell line; 12: mouse eye; 13: SK-RC-7 renal cancer cell line; 14: adult human brain. Gangliosides were separated in solvent 1 and visualized with resorcinol-HCl.
FIG. 4 densiometric tracings of thin layer chromatograms of gangliosides from melanomas and other cells. A: SK-MEL-28 melanoma cell line; B: SK-MEL-37 melanoma cell line; C: SK-MEL-13 melanoma cell line; D: melanoma (surgical specimen); E: adult human brain; F: Raji B-cell line; G: MOLT-4 cell line; H: SK-RC-7 renal cell line. The amount of G.sub.D3, as % of total ganglioside fraction, was calculated from the areas of the peaks and is indicated in each panel.
FIG. 5 inhibition of reactivity of AbR.sub.24 with SK-MEL-28 melanoma cells by acidic glycolipid fractions from a variety of cell lines and tissues. Assay: PA-MHA. : AbR.sub.24 control; : adult human spleen; : adult human liver; 0: teleost eye; : SK-RC-7 renal cancer cell line; : adult human brain; : MeWo melanoma cell line; : SK-MEL-29 melanoma cell line; : SK-MEL-37 melanoma cell line; : mouse eye; : MOLT-4 T-cell line; .DELTA.: melanoma (surgical specimen); : SK-MEL-28 melanoma cell line.
FIG. 6 glycolipid-mediated immune adherence (GMIA) assay using AbR.sub.24. Well A: AbR.sub.24 -reactive glycolipid isolated from SK-MEL-28 melanoma cell line; well B: G.sub.D3 ganglioside; well C: no ganglioside; well D: G.sub.M2 and G.sub.M3 ganglioside mixture. Antibody: AbR.sub.24 (1:1000).
FIG. 7 detection of G.sub.D3 ganglioside by AbR.sub.24 in GMIA assays. AbR.sub.24 dilutions are indicated in the figure.
FIG. 8 detection of G.sub.D3 ganglioside on TLC plates by reactivity with AbR.sub.24 and .sup.125 I-Protein A. Right side: gangliosides visualized with resorcinol-HCl reagent; left side: gangliosides reacting with AbR.sub.24 and 125I-Protein A. Lane 1: AbR.sub.24 -reactive ganglioside; Lane 2: gangliosides extracted from adult human brain. Solvent 2.
FIG. 9 proposed pathways for the biosynthesis of gangliosides [modified after Yu and Ando (32)].